Since the inception of microbiology and genetic engineering, there has been a desire to be able to transfer traits from one organism to another.
Recombinant DNA technology has made it possible to develop molecular cloning vectors that allow expression of heterologous genes in prokaryotic cells (cells of lower life forms without a nucleus) and eukaryotic cells (cells of higher organisms). Bacterial systems provide important advantages such as ease of use and high expression but impose a number of limitations for synthesis of eukaryotic proteins. In particular, correct folding, proteolytic processing, glycosylation, secretion, and subunit assembly may not occur or may occur incorrectly in bacteria. For these and other reasons, eukaryotic cells are preferred for expression of eukaryotic genes.
It has also been difficult to obtain expression of certain genes, especially those of lower organisms in cells of higher organisms. This has been true for many reasons including the fact that gene control mechanisms are often significantly different.
It has been known that T7 and certain other bacteriophage RNA polymerases, for example SP6, GH1 and T3, are single subunit enzymes with high catalytic activity and strict promoter specificity, which have found wide application for in vitro synthesis of RNA and as the basis for high-level gene expression systems in Escherichia coli. One potential problem with use of a prokaryotic RNA polymerase in a eukaryotic cell, however, is the requirement for mRNA to be processed, capped, methylated, and polyadenylated. Another potential problem concerns the observation that eukaryotic RNA polymerases are produced in the cytoplasm but are transported and subsequently localized in the nucleus. A system using T7 or other bacteriophage RNA polymerases would require localization of the enzyme in the cytoplasm to act on foreign genes contained within the vector also localized in the cytoplasm. Therefore, a transient system which could utilize the advantages of a bacteriophage RNA polymerase, which could function in an eukaryotic environment to facilitate the expression of a foreign gene, and which is simple, widely applicable, and highly efficient, is desirable.